The need for radioprotective agents in protecting against the harmful effects of exposure to environmental radiation and cancer radiation therapy is well recognized. At present there are few agents available for protection of human tissues from the damaging effects of ionizing radiation, occurring either as a side effect of the treatment of various cancers, or through intentional or accidental exposure from other sources. Amifostine is the only drug currently approved by the federal Food and Drug Administration for the protection of normal tissue from the disruptive effects of cancer chemotherapy and radiation. An active metabolite of the drug is taken up by cells, where it scavenges the damaging free oxygen radicals caused by exposure to IR, UV or cancer chemotherapy. Other antioxidants have been used, but they all have very limited effectiveness. Polyamino acid compounds in various forms have been claimed as radioprotective agents in several patents, including U.S. Pat. Nos. 7,041,994, 6,114,394, 5,434,145, 5,354,782, and 5,217,964. Antiinflammatory drugs have also been used, but their effect is limited primarily to blocking the cascade of chemical reactions induced by cellular damage, rather than preventing or correcting the cellular damage that triggers the process.
Exposure to radiation increases the rate of damage to DNA in the cell nucleus. Sufficient DNA damage results in excessively prolonged, defective or arrested DNA transcription, leading to cell death. One approach to this problem therefore involves compositions and methods to preserve DNA transcription activity in the cell. Moreover, an agent with this property could also be used in cancer treatment to selectively protect normal cells from the damaging effects that radiation and antineoplastic agents have on all cells.
Ubiquitin is a small protein consisting of 76 amino acids that is important in the regulation of protein function in the cell. Ubiquitination and deubiquitination are enzymatically mediated processes by which ubiquitin is covalently bound to or unbound from a target protein. These processes have been implicated in the regulation of the cell cycle, apoptosis, the marking of transmembrane proteins such as receptors for removal, regulation of DNA transcription and repair, and other important functions. Proteins are targeted for degradation by the proteasome in the cell by being “tagged” with three or more ubiquitin molecules (polyubiquitination). Ubiquitin molecules are cleaved from a protein by deubiquitinating enzymes, which are cysteine proteases that operate through an active site thiol. The binding of a single ubiquitin molecule (monoubiquitination) does not generally target the monoubiquitinated protein for degradation. Rather, it may trigger activities such as DNA repair and gene silencing, among other functions. (Huang and D'Andrea, Mol. Cell Biol. 7:323-34 (2006)). Deubiquitination allows the ubiquitin to be recycled and restores the function of the deubiquitinated protein. There are approximately 95 different deubiquitinating enzymes in human cells (Huang et al., Nature Cell Biol. 8(4):339-47 (2006)). Among them, Ubiquitin Specific Protease 1 (USP1) has been found to regulate the repair of DNA damage induced by DNA crosslinking agents, which include agents such as mitomycin C (MMC), cisplatin, dipoxybutane (DEB), ionizing radiation (IR) and ultraviolet radiation (UV). USP1 has been shown to deubiquitinate monoubiquitinated FANCD2 (FANCD2-Ub), a protein that in monoubiquitinated form mediates DNA repair from the damage induced by the aforementioned agents. (Nijman et al., Molecular Cell 17:331-39 (2005)). USP1 also has been shown to deubiquitinate monoubiquitinated PCNA (PCNA-Ub), a protein that in monoubiquitinated form activates DNA translesion synthesis (TLS), a polymerase-mediated bypass of DNA lesions. (Huang, et al., Nature Cell Biol. 8(4):339-47 ((2006)).